The long term objectives of these studies is to understand the process by which chromaffin vesicles are formed. The specific aim of the proposed studies are to obtain information on the biogenesis and turnover of chromaffin vesicle and the effects of insulin, nerve growth factor, dexamethasone and cell stimulation on these processes. Primary cultures of adrenal medulla cells will be used to study the biosynthesis of specific soluble proteins (chromogranins and soluble dopamine-Beta-hydroxglase [S-DBH] which are released together with the catecholamines during secretion and the synthesis of a specific membrane protein (membrane-bound DBH [M-DBH] which is retained by the cell, and their incorporation into chromaffin vesicles. Since DBH is a component of both the soluble content and the membrane of the vesicle it serves as a marker for both of these fractions. Cells will be incubated with [35S] methionine and its incorporation into S-DBH, M-DBH and chromogranins of chromaffin vesicles and other subcellular fractions determined by immunoprecipitation with specific anti-sera followed by gel electrophoresis. Nascent chromaffin vesicles will be separated from mature vesicles by centrifugation through continuous sucrose density gradients. The turnover of the labeled proteins in mature vesicles will be studied as a function of time in culture. Reutilization of vesicle membranes for the formation of nascent vesicles will be examined in double isotope labeling experiments. Cells which have incorporated [35S]methionine into mature chromaffin vesicles will be treated with secretogues and after various periods of time exposed to [3H]methionine. The 35S/3H ratios in M-DBH, S-DBH and chromogranins present in nascent vesicles will be determined and compared to those of unstimulated cells. The effects of insulin, dexamethasone and nerve growth factor on the synthesis of the specific proteins, on their incorporation into nascent vesicles and on the rate of maturation of vesicles will be determined. These studies will provide basic information on the life cycle of the chromaffin vesicle. Since the adrenal medulla and sympathetic neuron have common features related to the synthesis, storage and secretion of catecholamines, it is anticipated that these studies will also be relavant to the synaptic vesicle of the sympathetic nerve and its physiological role in the regulation of the cardiovascular system and other autonomic functions.